A method for establishing an animal model of HER2-positive tumors using normal mice

Abstract

The present invention discloses a method for establishing an animal model of HER2-positive tumors using normal mice, comprising the following steps: (1) cell culture: culturing human gastric cancer cells NCI-N87 in a cell culture medium, and collecting cells in the logarithmic growth phase; (2) inoculation: inoculating human gastric cancer cells NCI-N87 into normal mice; (3) evaluation: 2 weeks after inoculation, the tumor diameter is ≥0.6 cm and the tumor formation rate is ≥80%, indicating that the animal model of HER2-positive tumors is successfully established; and the step of immunosuppressing normal mice is not included. The present invention uses NCI-N87 cells to directly inoculate normal mice to prepare an animal model, which is simple to operate, stable and reliable, and can greatly save experimental costs, overcoming many limitations of other current tumor vaccine animal models. The method for detecting the therapeutic efficacy or preventive efficacy of HER2-positive tumor vaccines described in the present invention can be used to screen tumor vaccine candidates in the laboratory, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for establishing an animal model of HER2 positive tumor using normal mice. Background Art

[0002] Malignant tumors are one of the main diseases that threaten human life and health. Traditional cancer treatments such as surgery, radiotherapy, and drug therapy all have certain limitations. Due to poor targeting, radiotherapy and drug therapy can easily damage normal human cells and produce adverse reactions. With the in-depth study of the molecular mechanisms of tumor occurrence and development and the rapid development of biotechnology, biological therapy has become the fourth mode of tumor treatment. Peptide tumor vaccines are vaccines composed of peptides from tumor cell-specific antigens, virus-related antigens, oncogenes or mutant proteins of tumor suppressor genes. With the assistance of adjuvants such as cytokines and chemokines, they can activate or enhance the body's own anti-tumor immunity, thereby killing and clearing tumor cells. Compared with traditional vaccines, peptide tumor vaccines have the advantages of easy synthesis and purification, high safety, and strong specificity. Therefore, peptide tumor vaccines are a hot topic in the research direction of tumor vaccines.

[0003] Human epidermal growth factor receptor 2 (HER2) is a 185kD protein encoded by the proto-oncogene HER2 / neu. It belongs to the epidermal growth factor receptor family and is a receptor tyrosine kinase that binds to the cell membrane surface and participates in the signal transduction pathway that leads to cell growth and differentiation. The HER2 gene is an important factor affecting tumor prognosis. Overexpression of HER2 / neu has been found in many types of tumor cells. Patients with HER2 overexpression have poor tumor prognosis and high recurrence rate. Therefore, HER2 is an ideal target for tumor treatment. Currently, a number of therapeutic drugs targeting HER2 (including small molecule drugs and monoclonal antibodies) have been launched on the market and have achieved good therapeutic effects.

[0004] At the same time, synthesizing epitope peptides on tumor-associated antigens of HER2 / neu as tumor vaccines, inoculating the body to produce specific cytotoxic T lymphocytes against HER2, and killing tumor cells is another important research direction for tumor treatment. At present, several peptide vaccines targeting HER2 have entered or completed the phase II clinical trial stage, and one has entered the phase III clinical trial stage. The results of relevant clinical trials have shown that peptide vaccines can stimulate and induce human CD4+T and CD8+T lymphocytes, significantly increase the levels of both in the body, and kill tumor cells.

[0005] However, it is difficult to evaluate the efficacy of tumor vaccines in animals. The difficulty lies in the lack of an ideal animal model for efficacy evaluation. Several existing animal models have their limitations and are difficult to be used for the efficacy evaluation of tumor vaccines: ① Traditional immunodeficient animals (nude mice or SCID mice) used to evaluate the efficacy of anti-tumor drugs cannot be used for the efficacy evaluation of tumor vaccines due to their immune system defects, because tumor vaccines achieve anti-tumor effects by activating the body's immune system; ② Normal mice are difficult to successfully inoculate human tumor cells due to immune rejection. Even if they can be inoculated with human tumor cells after drug immunosuppression, immunosuppressed mice are not suitable for evaluating the efficacy of tumor vaccines; ③ Although immune reconstruction mice can reconstruct the immune system in immunodeficient animals, the operation is cumbersome, the stability is poor, and the animals are prone to die due to immune rejection; ④ Transgenic mice are currently the most suitable animal model for evaluating the efficacy of tumor vaccines, but the cost is high, which limits their use. Therefore, a method for establishing an animal model for evaluating the efficacy of tumor vaccines that is easy to operate and economical is needed. Summary of the invention

[0006] The purpose of the present invention is to provide a method for establishing an animal model of HER2-positive tumors using normal mice, so as to solve the problems raised in the above-mentioned background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for establishing an animal model of HER2-positive tumor using normal mice comprises the following steps:

[0009] (1) Cell culture: Human gastric cancer cells NCI-N87 were cultured in a cell culture medium, and cells in the logarithmic growth phase were collected;

[0010] (2) Inoculation: Human gastric cancer cells NCI-N87 were inoculated into normal mice;

[0011] (3) Evaluation: 2 weeks after inoculation, the tumor diameter was ≥0.6 cm and the tumor formation rate was ≥80%, indicating that the animal model of HER2-positive tumors was successfully established;

[0012] The method does not include the step of immunosuppressing normal mice.

[0013] As a further embodiment of the present invention: the HER2-positive tumor is a gastric cancer tumor.

[0014] As a further solution of the present invention: In step (2), human gastric cancer cell NCI-N87 is 1.25×10 5 -1×10 6 Normal mice were inoculated with a concentration range of 100 / 100 mice.

[0015] As a further embodiment of the present invention, human gastric cancer cells NCI-N87 were cultured at 5×10 5 Normal mice were inoculated with a concentration of 1000 / mouse.

[0016] As a further embodiment of the present invention: the normal mice are mice with normal immune function.

[0017] As a further embodiment of the present invention: the mice with normal immune function are Balb / c mice.

[0018] The present invention also provides a method for detecting the therapeutic efficacy of a HER2-positive tumor vaccine for non-diagnostic purposes, comprising the following steps:

[0019] (1) Model establishment: HER2-positive human gastric cancer cells NCI-N87 were cultured at 5×10 5 Inoculate normal mice to establish a transplanted tumor animal model;

[0020] (2) Immunization: immunizing the animal model with transplanted tumors in step (1) with HER2-positive tumor vaccines at different concentrations;

[0021] (3) Record: After immunization, the tumor growth was recorded every day, the long and short diameters of the tumor were measured, the tumor volume was calculated, and the weight of the mice was recorded at the same time;

[0022] (4) Evaluation: 28 days after the last immunization, the tumor inhibition rate of tumor vaccines at different concentrations was calculated to evaluate the therapeutic efficacy of the tumor vaccine.

[0023] The present invention also provides a method for detecting the preventive efficacy of a HER2-positive tumor vaccine for non-diagnostic purposes, comprising the following steps:

[0024] (1) Immunization: Normal mice were first immunized with different concentrations of HER2-positive tumor vaccines;

[0025] (2) Model establishment: HER2-positive human gastric cancer cells NCI-N87 were cultured at 5×10 5 Inoculate mice immunized with tumor vaccine in step (1) to establish a transplanted tumor animal model;

[0026] (3) Record: After immunization, the tumor growth was recorded every day, the long and short diameters of the tumor were measured, the tumor volume was calculated, and the weight of the mice was recorded at the same time;

[0027] (4) Evaluation: 28 days after the last immunization, the incidence and tumor inhibition rate of tumor vaccines at different concentrations were calculated to evaluate the preventive efficacy of tumor vaccines.

[0028] Another object of the present invention is to provide the use of human gastric cancer cell NCI-N87 in establishing an animal model of HER2-positive tumor using normal mice, wherein the use does not include immunosuppression of normal mice.

[0029] As a further embodiment of the present invention: the HER2-positive tumor is a gastric cancer tumor.

[0030] As a further embodiment of the present invention: the normal mice are mice with normal immune function.

[0031] As a further embodiment of the present invention: the mice with normal immune function are Balb / c mice.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention provides a method for establishing an animal model of HER2-positive tumors, and screens out human gastric cancer cells NCI-N87 that can be directly inoculated into normal mice to produce tumors, which is suitable for use as an animal model for preclinical in vivo anti-tumor activity evaluation. This is completely unexpected, because the prior art only records the use of human gastric cancer cells NCI-N87 to inoculate nude mice or severely immunodeficient mice to prepare xenograft tumor animal models (see: Zhang C, et al. Establishing aperitoneal dissemination xenograft mouse model for survival outcome assessment of experimental gastric cancer. J Surg Res. 2013 Jun 15; 182(2): 227-34; YS Lee, et al. Tumor dosimetry for I-131 trastuzumab therapy in a Her2+ NCIN87 xenograft mouse model using the Siemens SYMBIA E gamma camera with apinhole collimator. Journal of Instrumentation, 2015, 10(7)), and there is no literature report on the use of human gastric cancer cells NCI-N87 to inoculate normal mice to cause tumors. The animal model prepared by directly inoculating normal mice with human gastric cancer cells NCI-N87 is simple to operate, stable and reliable, and can greatly save experimental costs, overcoming many limitations of other current tumor vaccine animal models. The therapeutic effect of tumor vaccines can be evaluated by first inoculating tumor cells and then immunizing the vaccine, and measuring the inhibition rate of tumor volume; or first immunizing the vaccine and then inoculating the tumor to evaluate the preventive effect of the vaccine on tumors. In addition, the method for detecting the therapeutic efficacy or preventive efficacy of HER2-positive tumor vaccines described in the present invention can be used to screen tumor vaccine candidates in the laboratory, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the HPLC spectrum of solid phase synthesized E75.

[0035] Figure 2 This is a graph showing the tumor volume growth curve of the NCI-N87 animal model treated with E75.

[0036] Figure 3 This is a curve of body weight changes in the NCI-N87 animal model treated with E75.

[0037] Figure 4This is the preventive effect of E75 on the NCI-N87 animal model: a graph showing changes in tumor incidence.

[0038] Figure 5 This is the preventive effect of E75 on the NCI-N87 animal model: tumor volume change curve.

[0039] Figure 6 This is the preventive effect of E75 on the NCI-N87 animal model: body weight change curve. DETAILED DESCRIPTION

[0040] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0041] The inventors have conducted a lot of research work, hoping to establish a convenient and practical animal model for evaluating the effect of tumor vaccines. During the research process, they unexpectedly found that human gastric cancer cells NCI-N87 can be directly inoculated into normal mice to form a transplanted tumor animal model without immunosuppression of normal mice, which can be used to screen tumor vaccines or anticancer drugs. The specific research process is as follows:

[0042] In the following examples, tumor vaccine peptide E75 was selected to evaluate the effect of the established animal model on the efficacy of HER2-positive tumor vaccines. E75 is the most studied peptide tumor vaccine and has entered Phase III clinical studies. Its clinical efficacy against HER2-positive tumors has been widely recognized.

[0043] Example 1 Screening of human HER2-positive tumor cells

[0044] Ten HER2-positive human tumor cell lines were selected, including 7 breast cancer cell lines: SK-BR-3, MDA-MB-453, BT-474, HCC1008, HCC1569, HCC2218, and HCC1419, 1 ovarian cancer cell line: SKOV-3, and 2 gastric cancer cell lines: SGC-7901 and NCI-N87. All cells were purchased from ATCC.

[0045] Cell culture: The above cell lines were revived from cryopreserved tubes, cultured in RPMI 1640 medium, and subcultured until the cells were in good growth state for later use.

[0046] Inoculation: 60 8-week-old Balb / c mice were randomly divided into 10 groups, with 6 mice in each group. After one week of adaptation, they were inoculated with the above 10 tumor cells. The inoculation cell concentration was 5×106 Each mouse was inoculated with 0.1 ml of the drug at the axilla of the left forelimb.

[0047] After tumor inoculation, tumor growth was observed and recorded daily. Two weeks after inoculation, all animals were killed to terminate the experiment and evaluate the experimental results. Tumor diameter ≥ 0.6 cm and tumor formation rate ≥ 80% were used as the criteria for successful tumor transplantation.

[0048] Results: Among the 10 groups of animals, only human gastric cancer cell NCI-N87 could be successfully transplanted into normal Balb / c mice, and all 6 mice in the NCI-N87 group successfully formed tumors (tumor formation rate was 100%). The other 9 types of human tumor cells could not be successfully transplanted into Balb / c (tumor formation rate was 0). Therefore, human gastric cancer cell NCI-N87 can successfully cause tumors in Balb / c mice, and the tumor-bearing animals can be used as a model for evaluating the efficacy of HER2-positive tumor vaccines.

[0049] Example 2 Optimization of the cell inoculation amount for mice transplanted with human gastric cancer cells NCI-N87

[0050] In order to establish the best NCI-N87 animal model, the inoculation amount of human gastric cancer cell NCI-N87 was optimized.

[0051] Twenty-four 8-week-old Balb / c mice were randomly divided into 4 groups, with 6 mice in each group. After one week of adaptation, they were inoculated with a concentration of 1.25×10 6 / ml, 2.5×10 6 / ml, 5×10 6 / ml, 1×10 7 Each animal was inoculated with 0.1 ml of human gastric cancer cell NCI-N87 at the axilla of the left forelimb. After tumor inoculation, the tumor growth was observed and recorded daily. Two weeks after inoculation, all animals were killed, the experiment was terminated, and the experimental results were evaluated.

[0052] Result: 1.25×10 6 Three animals in the group with 2.5×10 / ml developed tumors, with a tumor formation rate of 50%. 6 / ml, 5×10 6 / ml, 1×10 7 All animals in the 5×10 / ml group had tumor growth, with a tumor formation rate of 100% and a tumor diameter between 6 and 10 mm, which is suitable for evaluating the efficacy of tumor drugs or vaccines. 6 The tumor diameter of the group with cells / ml was about 8mm, which was the optimal inoculation amount.

[0053] Example 3 Solid Phase Synthesis of Tumor Vaccine Peptide E75

[0054] E75 was commissioned by Shanghai Aobo Biotechnology Co., Ltd. to synthesize the peptide using conventional solid phase technology. The purity of the synthesized peptide is >98%. Figure 1 .

[0055] Evaluation of the efficacy of tumor vaccine therapy using the NCI-N87 animal model

[0056] Inoculation: 40 8-week-old female Balb / c mice were used in this study. Human gastric cancer cells NCI-N87 in the logarithmic growth phase were collected and prepared into 5×10 6 0.1 ml of the cell suspension was seeded in the left armpit of the mouse; the tumor growth was observed every day, and immunotherapy was started when the tumor grew to about 8 mm in diameter.

[0057] Immunization: 40 mice were randomly divided into 4 groups, 10 mice in each group, which were negative control group (PBS) and E75 low, medium and high dose groups. E75 was mixed and emulsified with Freund's incomplete adjuvant (FIA) at 1:1 for 30 minutes at 100, 300 and 1000 μg / ml, and PBS was mixed and emulsified with Freund's incomplete adjuvant (FIA) at 1:1 for 30 minutes as negative control. After emulsification, each group of animals was immunized at 200 μl / mouse, so that the final E75 inoculation amount of each group of animals was 0, 10, 30 and 100 μg / mouse, respectively. The inoculation method was subcutaneous injection, and the inoculation site was the right side of the armpit. The drug was administered once a week for a total of three immunizations.

[0058] Record: The tumor occurrence of each group of animals was recorded every day. The long and short diameters of the tumor were measured with a vernier caliper every other day. The tumor volume was calculated using the following formula, and the body weight was recorded.

[0059] TV=1 / 2×a×b 2 , where a and b represent the length and width respectively.

[0060] Evaluation: Four weeks after the last immunization, all mice were killed, the experiment was terminated, and the tumor inhibition rate was calculated for evaluation.

[0061] Tumor inhibition rate (%) = (tumor volume of control group - tumor volume of experimental group) / tumor volume of control group × 100%. SPSS software was used for statistical analysis and one-way analysis of variance was used for comparison. P < 0.05 was considered statistically significant.

[0062] Results: After 28 days of treatment, the tumor volumes of the low, medium and high dose E75 groups were significantly reduced compared with the negative control group (P < 0.05), and the tumor inhibition rates were 29.6%, 47.6% and 41.4%, respectively. Figure 2), proving that E75 can significantly reduce tumor volume and that the established animal model can be used to evaluate the therapeutic efficacy of E75. At the same time, the weight of mice in each group increased slightly, with no significant difference between the groups, indicating that E75 is safe and that the animal model can also be used to evaluate the safety of tumor vaccines ( Figure 3 ).

[0063] Example 4 Evaluation of the preventive efficacy of tumor vaccines using the NCI-N87 animal model

[0064] Immunization: 8-week-old female Balb / c mice were randomly divided into 4 groups, 15 in each group, including negative control group (PBS) and E75 low, medium and high dose groups. E75 vaccine was mixed with Freund's incomplete adjuvant (FIA) at 1:1 for 30 minutes at 100, 300 and 1000 μg / ml, and PBS was mixed with Freund's incomplete adjuvant (FIA) at 1:1 for 30 minutes as negative control. After emulsification, each group of animals was immunized at 200 μl / mouse, so that the final E75 inoculation amount of each group of animals was 0, 10, 30 and 100 μg / mouse, respectively. The vaccination method was subcutaneous injection, and the vaccination site was the right armpit. The vaccine was administered once a week for a total of three immunizations.

[0065] Inoculation: One week after the third immunization, human gastric cancer cells NCI-N87 in the logarithmic growth phase were collected and prepared into 5×10 6 0.1 ml of the cell suspension was inoculated into the axilla of the left forelimb of the mouse.

[0066] Record: The tumor occurrence of each group of animals was recorded every day. After the tumor grew, the long and short diameters of the tumor were measured with a vernier caliper every other day to calculate the tumor volume;

[0067] TV=1 / 2×a×b 2 , where a and b represent the major diameter and minor diameter, respectively, and the weight of animals in each group was recorded.

[0068] Evaluation: Four weeks after tumor inoculation, all mice were killed, the experiment was terminated, and the tumor incidence and tumor inhibition rate were calculated and evaluated.

[0069] Tumor incidence (%) = number of animals with tumors in each group / total number of animals in each group × 100%

[0070] Tumor inhibition rate (%) = (average tumor volume of the control group - average tumor volume of the experimental group) / average tumor volume of the control group × 100%.

[0071] Data processing: SPSS software was used for statistical analysis, and one-way analysis of variance was used for comparison. P < 0.05 was considered statistically significant.

[0072] Results: 25 days after tumor inoculation, the tumor incidence rates in the negative control group, E75 low-dose, medium-dose, and high-dose groups were 93.3%, 73.3%, 46.7%, and 53.3%, respectively. The tumor incidence rates in the medium-dose and high-dose groups were significantly lower than those in the negative control group (P < 0.05). Figure 4 ), showing that E75 significantly reduced the incidence of NCI-N87 tumors, and compared with the control group, the tumor volume in each treatment group was significantly inhibited ( Figure 5 ), proving that the established NCI-N87 animal model can be used to evaluate the preventive efficacy of E75. At the same time, the weight of mice in each group increased slightly, and there was no significant difference between the groups, indicating that E75 is safe. This animal model can also be used to evaluate the safety of tumor vaccines ( Figure 6 ).

[0073] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0074] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for establishing an animal model of HER2-positive tumors using normal mice, characterized in that: The following steps are involved: (1) Cell culture: Human gastric cancer cells NCI-N87 were cultured in a cell culture medium, and cells in the logarithmic growth phase were collected; (2) Inoculation: Human gastric cancer cells NCI-N87 were inoculated into normal mice at the axilla of the left forelimb; (3) Evaluation: 2 weeks after inoculation, the tumor diameter was ≥0.6 cm and the tumor formation rate was ≥80%, indicating that the animal model of HER2-positive tumors was successfully established; The method does not include the step of immunosuppressing normal mice; the normal mice are Balb / c mice with normal immune function.

2. The method for establishing an animal model of HER2-positive tumor using normal mice according to claim 1, characterized in that: The HER2-positive tumor is a gastric cancer tumor.

3. The method for establishing an animal model of HER2-positive tumor using normal mice according to claim 1, characterized in that: In step (2), human gastric cancer cells NCI-N87 were isolated at 1.25×10 5 -1×10 6 Normal mice were inoculated with a concentration range of 100 / 100 mice.

4. The method for establishing an animal model of HER2-positive tumor using normal mice according to claim 3, characterized in that: Human gastric cancer cells NCI-N87 were cultured at 5×10 5 Normal mice were inoculated with a concentration of 1000 / mouse.

5. A method for detecting the therapeutic efficacy of a HER2-positive tumor vaccine for non-diagnostic purposes, characterized in that: The following steps are involved: (1) Model establishment: HER2-positive human gastric cancer cells NCI-N87 were cultured at 5×10 5 Inoculate normal mice to establish a transplanted tumor animal model; (2) Immunization: immunizing the animal model with transplanted tumors in step (1) with HER2-positive tumor vaccines at different concentrations; (3) Record: After immunization, the tumor growth was recorded every day, the long and short diameters of the tumor were measured, the tumor volume was calculated, and the weight of the mice was recorded at the same time; (4) Evaluation: 28 days after the last immunization, the tumor inhibition rate of tumor vaccines at different concentrations was calculated to evaluate the therapeutic efficacy of the tumor vaccine; The normal mice are Balb / c mice with normal immune function, and the inoculation site is the axilla of the left forelimb.

6. A method for detecting the preventive efficacy of a HER2-positive tumor vaccine for non-diagnostic purposes, characterized in that: The following steps are involved: (1) Immunization: Normal mice were first immunized with different concentrations of HER2-positive tumor vaccines; (2) Model establishment: HER2-positive human gastric cancer cells NCI-N87 were cultured at 5×10 5 Inoculate mice immunized with tumor vaccine in step (1) to establish a transplanted tumor animal model; (3) Record: After immunization, the tumor growth was recorded every day, the long and short diameters of the tumor were measured, the tumor volume was calculated, and the weight of the mice was recorded at the same time; (4) Evaluation: 28 days after the last immunization, the incidence and tumor inhibition rate of tumor vaccines at different concentrations were calculated to evaluate the preventive efficacy of tumor vaccines; The normal mice are Balb / c mice with normal immune function, and the inoculation site is the axilla of the left forelimb.

7. Use of human gastric cancer cell NCI-N87 in establishing an animal model of HER2-positive tumor using normal mice, characterized in that: The use does not include immunosuppression of normal mice, the normal mice are Balb / c mice with normal immune function, and the vaccination site is the axilla of the left forelimb.

8. The use of human gastric cancer cell NCI-N87 according to claim 7 in establishing an animal model of HER2-positive tumor using normal mice, characterized in that: The HER2-positive tumor is a gastric cancer tumor, and the normal mouse is a Balb / c mouse with normal immunological function.

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